Journal: PLoS ONE

Article Title: Macrophages and Dendritic Cells Emerge in the Liver during Intestinal Inflammation and Predispose the Liver to Inflammation

doi: 10.1371/journal.pone.0084619

Figure Lengend Snippet: ( A ) FACS analysis of PDCA-1 + CD11b − CD11c int pDCs from the livers of WT (left column) mice. We also analyzed CD11b + CD11c − Mφs from the livers of ConA-treated (middle) and IL-10 −/− (right) mice, respectively. Dead cells were excluded with 7AAD staining. ( B ) Proliferation of naïve CFSE-labeled splenic CD4 + T cells from OT-II mice, and co-cultured WT pDCs, ConA Mφs, or IL-10 −/− Mφs in the presence of OVA. Dead cells were excluded with 7AAD staining and CD4 + T cells gated on CD3 + CD4 + cells are shown (B and C). Data are representative of three independent experiments. ( C ) Intracellular IFN-γ and IL-17A expression in CD4 + T cells co-cultured with WT pDCs, ConA Mφs, or IL-10 −/− Mφs in the presence of OVA. Data are representative of three independent experiments. ( D ) Proportion of IFN-γ + IL-17A − , IFN-γ − IL-17A + , and IFN-γ + IL-17A + cells among the Th cell population. ( E ) Cytokine concentrations in the culture supernatant of OT-II CD4 + T cells that were co-cultured with WT pDCs or ConA Mφs. Data are representative of three independent experiments. Each experiment was performed using duplicate samples. * P <0.05.

Article Snippet: Enriched naïve CD4 + splenocytes obtained from OT-II mice were sorted using a CD4 + CD62L + T Cell Isolation Kit II (Miltenyi Biotech, Auburn, CA, USA) and labeled with 1 mM CFSE (Molecular Probes, Eugene, OR, USA) for 10 min at 37°C, followed by the addition of 1.0 ml of FCS for 2 min and washed three times in PBS.

Techniques: Staining, Labeling, Cell Culture, Expressing

Journal: PLoS ONE

Article Title: Macrophages and Dendritic Cells Emerge in the Liver during Intestinal Inflammation and Predispose the Liver to Inflammation

doi: 10.1371/journal.pone.0084619

Figure Lengend Snippet: ( A ) Numbers of hepatic mononuclear cells. Data are presented as the mean ± SEM for each group. RAG-2 −/− mice ( n = 4) and RAG-2 −/− LP CD4 + mice ( n = 4). ( B ) Representative data from flow cytometry analysis of Th cells in each organ. Dead cells were excluded by 7AAD staining. ( C ) Numbers of hepatic CD3 + CD4 + Th cells and non-T cells. ( D ) Representative data from flow cytometry analysis of pDCs and Mφs in the liver of each experimental group. Dead cells were excluded using 7AAD staining. Scatter plots for CD11b − CD11c int and CD11b + CD11c − cells are shown in the middle and bottom rows, respectively. ( E ) Proportion of PDCA-1 + CD11b − CD11c int pDCs and F4/80 + CD11b + CD11c − Mφs among whole mononuclear cells. Data are representative of three independent experiments. Values are presented as the mean ± SEM from seven mice in each group. * P <0.05, ** P <0.01, *** P <0.005.

Article Snippet: Enriched naïve CD4 + splenocytes obtained from OT-II mice were sorted using a CD4 + CD62L + T Cell Isolation Kit II (Miltenyi Biotech, Auburn, CA, USA) and labeled with 1 mM CFSE (Molecular Probes, Eugene, OR, USA) for 10 min at 37°C, followed by the addition of 1.0 ml of FCS for 2 min and washed three times in PBS.

Techniques: Flow Cytometry, Staining

Journal: PLoS ONE

Article Title: Macrophages and Dendritic Cells Emerge in the Liver during Intestinal Inflammation and Predispose the Liver to Inflammation

doi: 10.1371/journal.pone.0084619

Figure Lengend Snippet: ( A ) H&E specimens of the colon taken from mice treated with water (left) or 2% DSS (right). Magnification, ×40 ( B ) H&E specimens of livers from mice treated with water (left) and DSS (right). Magnification, ×100 ( C ) The number of liver mononuclear cells from water- and DSS-treated mice. ( D ) The absolute number of PDCA-1 + CD11b − CD11c int pDCs and CD11b + CD11c − Mφs among whole mononuclear cells. Data are representative of three independent experiments. ( E ) Levels of mRNA transcripts for IFN-γ, TNF, and IL-6 in the liver. Values are presented as the mean ± SEM for each group ( n = 4, water-treated group; n = 5, DSS-treated group). * P <0.05. N.S., no significant difference. ( F–H ) Hepatic DSS Mφs induce a Th1 inflammatory response. ( F ) Proliferation of naïve CFSE-labeled splenic CD4 + T cells. ( G ) Intracellular IFN-γ and IL-17A expression in naïve CFSE-unlabeled splenic CD4 + T cells from OT-II mice that were co-cultured with or without hepatic Mφs from DSS-treated WT mice in the presence of OVA. Dead cells were excluded with 7AAD staining, and CD4 + T cells gated on CD3 + CD4 + cells are shown. Data are representative of two independent experiments. ( H ) Representative cytokine concentrations in culture supernatants from two independent experiments. Each experiment was performed using duplicate samples.

Article Snippet: Enriched naïve CD4 + splenocytes obtained from OT-II mice were sorted using a CD4 + CD62L + T Cell Isolation Kit II (Miltenyi Biotech, Auburn, CA, USA) and labeled with 1 mM CFSE (Molecular Probes, Eugene, OR, USA) for 10 min at 37°C, followed by the addition of 1.0 ml of FCS for 2 min and washed three times in PBS.

Techniques: Labeling, Expressing, Cell Culture, Staining

Journal: PLoS ONE

Article Title: Neutrophil Gelatinase-Associated Lipocalin Increases HLA-G + /FoxP3 + T-Regulatory Cell Population in an In Vitro Model of PBMC

doi: 10.1371/journal.pone.0089497

Figure Lengend Snippet: A) Flow cytometry analysis of surface HLA-G on CD4 + and CD8 + cells. PBMCs were treated with increasing concentrations of NGAL:Enterobactin:Iron (40 ng/ml, 80 ng/ml, 160 ng/ml, and 320 ng/ml). A dose-dependent rise in the percentage of CD4 + HLA-G + cells was evident. The percentage of CD8 + HLA-G + cells did not show any marked changes. Data are representative of 8 independent experiments. The number in each quadrant represents the percentage of the total population. B) Data are expressed as percentages of CD4 + HLA-G + cells. Means ± SD; n = 8. * p<0.05.

Article Snippet: Detection of CD4+ CD25+ FoxP3+ regulatory T cells on PBMC was performed with the Treg Detection Kit, CD4/CD25/FoxP3 (Miltenyi, Bergish Gladbach, Germany) as described in the manufacturer's protocol.

Techniques: Flow Cytometry

Journal: PLoS ONE

Article Title: Neutrophil Gelatinase-Associated Lipocalin Increases HLA-G + /FoxP3 + T-Regulatory Cell Population in an In Vitro Model of PBMC

doi: 10.1371/journal.pone.0089497

Figure Lengend Snippet: Flow cytometry analysis of HLA-G expression on CD4 + cells. Iron treatment (without NGAL) did not increase the percentage of HLA-G + cells. Treatment with the NGAL:enterobactin complex (without iron) reduced HLA-G expression in contrast to stimulation with NGAL:Enterobactin:Iron. Incubation with anti-NGAL antibody reduced the percentage of HLAG + cells. Data are representative of 8 independent experiments. The number in each quadrant represents the percentage of the total population. B) Data are expressed as percentages of CD4 + HLA-G + cells. Means ± SD; n = 8. * p<0.05.

Article Snippet: Detection of CD4+ CD25+ FoxP3+ regulatory T cells on PBMC was performed with the Treg Detection Kit, CD4/CD25/FoxP3 (Miltenyi, Bergish Gladbach, Germany) as described in the manufacturer's protocol.

Techniques: Flow Cytometry, Expressing, Incubation

Journal: PLoS ONE

Article Title: Neutrophil Gelatinase-Associated Lipocalin Increases HLA-G + /FoxP3 + T-Regulatory Cell Population in an In Vitro Model of PBMC

doi: 10.1371/journal.pone.0089497

Figure Lengend Snippet: A). The percentage of CD4 + /CD25 + /FoxP3 + cells in PHA-activated PBMCs after stimulation with increasing concentrations of NGAL: Enterobactin:Iron (40 ng/ml, 80 ng/ml, 160 ng/ml, and 320 ng/ml), raised in a dose dependent manner. Data are representative of 8 independent experiments. The number in each quadrant represents the percentage of CD25 + /FoxP3 + cells on gated CD4 + cells of the total population. B) Data are expressed as percentages of CD4 + /CD25 + /FoxP3 + cells. Means ± SD; n = 8. *p<0.05.

Article Snippet: Detection of CD4+ CD25+ FoxP3+ regulatory T cells on PBMC was performed with the Treg Detection Kit, CD4/CD25/FoxP3 (Miltenyi, Bergish Gladbach, Germany) as described in the manufacturer's protocol.

Techniques:

Journal: PLoS ONE

Article Title: Neutrophil Gelatinase-Associated Lipocalin Increases HLA-G + /FoxP3 + T-Regulatory Cell Population in an In Vitro Model of PBMC

doi: 10.1371/journal.pone.0089497

Figure Lengend Snippet: Flow cytometry analysis of CD25 + /FoxP3 + expression on CD4 + cells in PHA-activated PBMCs. Iron treatment (without NGAL) did not increase the percentage of CD25 + /FoxP3 + cells. Treatment with NGAL:enterobactin complex (without iron) reduced the percentage of CD25 + /FoxP3 + cells in contrast to stimulation with NGAL:Enterobactin:Iron. Incubation with anti-NGAL antibody reduced the percentage of CD25 + /FoxP3 + cells. Data are representative of 8 independent experiments. The number in each quadrant represents the percentage of CD25+foxP3 cells on gated CD4 + cells of the total population. B) Data are expressed as percentages of CD4+/CD25+/FoxP3+ cells. Means ± SD; n = 8. * p<0.05.

Article Snippet: Detection of CD4+ CD25+ FoxP3+ regulatory T cells on PBMC was performed with the Treg Detection Kit, CD4/CD25/FoxP3 (Miltenyi, Bergish Gladbach, Germany) as described in the manufacturer's protocol.

Techniques: Flow Cytometry, Expressing, Incubation

Journal: PLoS ONE

Article Title: Neutrophil Gelatinase-Associated Lipocalin Increases HLA-G + /FoxP3 + T-Regulatory Cell Population in an In Vitro Model of PBMC

doi: 10.1371/journal.pone.0089497

Figure Lengend Snippet: Flow cytometry analysis of HLA-G + /FoxP3 + expression on CD4 + cell PHA-activated PBMCs. PBMCs were treated with increasing concentrations of NGAL:Enterobactin:Iron (40 ng/ml, 80 ng/ml, 160 ng/ml, and 320 ng/ml). A dose-dependent raise in the percentage of HLA-G + /FoxP3 + cells was evident. Data are representative of 8 independent experiments. The number in each quadrant represents the percentage of HLA-G + /FoxP3 + cells on gated CD4 + cells of the total population. B) Data are expressed as percentages of CD4 + /HLA-G + /FoxP3 + cells. Means ± SD; n = 8. * p<0.05.

Article Snippet: Detection of CD4+ CD25+ FoxP3+ regulatory T cells on PBMC was performed with the Treg Detection Kit, CD4/CD25/FoxP3 (Miltenyi, Bergish Gladbach, Germany) as described in the manufacturer's protocol.

Techniques: Flow Cytometry, Expressing